CN117924527A - Wheat bran-based anti-inflammatory active polysaccharide and preparation method and application thereof - Google Patents
Wheat bran-based anti-inflammatory active polysaccharide and preparation method and application thereof Download PDFInfo
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- CN117924527A CN117924527A CN202311727016.8A CN202311727016A CN117924527A CN 117924527 A CN117924527 A CN 117924527A CN 202311727016 A CN202311727016 A CN 202311727016A CN 117924527 A CN117924527 A CN 117924527A
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- wheat bran
- polysaccharide
- inflammatory active
- active polysaccharide
- inflammatory
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- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses an anti-inflammatory active polysaccharide based on wheat bran, a preparation method and application thereof, wherein activated paecilomyces cicadae is utilized to prepare the anti-inflammatory active polysaccharide through solid fermentation of wheat bran, the wheat bran is subjected to solid fermentation based on vigorous fertility and stronger bioconversion capability, and then water extraction and alcohol precipitation are carried out to obtain the fermented wheat bran polysaccharide, the anti-inflammatory active polysaccharide consists of mannose, glucuronic acid, glucose and galactose, the weight average molecular weight distribution range is 1.3 multiplied by 10 2Da～2.93×105 Da, the influence of the anti-inflammatory active polysaccharide on ulcerative colitis of mice is explored through in vivo animal experiments, and the research result shows that FWBP has more remarkable anti-inflammatory effect.
Description
Technical Field
The invention belongs to the technical field of biological fermentation, and particularly relates to anti-inflammatory active polysaccharide based on wheat bran, and a preparation method and application thereof.
Background
Wheat bran is a main byproduct in wheat flour milling production, is rich in dietary fiber, starch, protein, vitamin E, lignin and other nutritional ingredients, and has high application value. The wheat bran contains up to 35% -60% of dietary fiber, 10% -18% of protein and 10% -15% of starch, and is an ideal dietary fiber source.
Dietary fiber is considered as an important ingredient of health-promoting foods, and is classified into soluble dietary fiber and insoluble dietary fiber according to its water solubility. The soluble dietary fiber mainly contains arabinoxylan and glucan, and the monosaccharide composition comprises xylose, glucose and arabinose, and the structure contains more hydrophilic groups, so that the soluble dietary fiber can be utilized by probiotic glycolysis in intestinal tracts, is considered to play a more important physiological role, is beneficial to maintaining stable blood sugar, reducing blood cholesterol level, regulating intestinal flora and reducing colorectal cancer risk. The insoluble dietary fiber can absorb toxic substances in food, and has effects of loosening bowel to relieve constipation and preventing constipation.
The ratio of soluble dietary fiber to insoluble dietary fiber has a significant impact on the physiological function of wheat bran dietary fiber, and it is generally considered that the mass ratio is 1:3 is preferable. The dietary fiber in the wheat bran is mainly insoluble dietary fiber, and the content of the soluble dietary fiber is only about 3%. Therefore, the content of the soluble dietary fiber of the wheat bran can be increased through physical, chemical or biological technology, the biological activity effect of the soluble dietary fiber of the wheat bran is improved, and the application and development of the wheat bran active polysaccharide in the biological and medical fields are widened.
Paecilomyces cicadae Paecilomyces cicadae (Miquel.) Samson is a traditional rare Chinese medicine of Cordyceps cicadae and also called Cordyceps cicadae, belongs to a parasitic medicinal fungus, is an insect-fungus complex formed by parasitizing Paecilomyces cicadae on a certain homoptera cicadae insect nymph, and contains various active ingredients such as protein, polysaccharide, nucleoside substances and the like. The cordyceps sobolifera polysaccharide has outstanding surface in the aspects of easing pain, reducing blood sugar, resisting tumor, protecting kidney, regulating immunity and the like.
In recent years, microbial solid state fermentation has received increasing attention to the preparation of active polysaccharides. The microbial fermentation is utilized to degrade or convert the nutritional ingredients such as protein, starch and the like in the raw materials, and the insoluble dietary fiber is converted into the soluble dietary fiber, so that the biological activity of the insoluble dietary fiber is improved. The microbial high-solid fermentation preparation process has the following potential advantages: ① The solid content of the wheat bran substrate is improved, the productivity of unit equipment is increased, and the energy consumption in the subsequent drying process is reduced; ② The water consumption is reduced, the cost is saved, and the wastewater discharge is reduced; ③ Improving the biological activity of the dietary fiber of the wheat bran is an excellent method for modifying the dietary fiber in the wheat bran.
Therefore, development of the anti-inflammatory active polysaccharide prepared by using the paecilomyces cicadae solid-state fermentation wheat bran has important significance in overcoming a plurality of problems existing in natural wheat bran dietary fibers.
Disclosure of Invention
This section is intended to outline some aspects of embodiments of the application and to briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section as well as in the description of the application and in the title of the application, which may not be used to limit the scope of the application.
The present invention has been made in view of the above and/or problems occurring in the prior art.
Therefore, the invention aims to overcome the defects in the prior art and provide the wheat bran-based anti-inflammatory active polysaccharide.
In order to solve the technical problems, the invention provides the following technical scheme: the anti-inflammatory active polysaccharide is prepared by solid state fermentation of wheat bran by using paecilomyces cicadae (ISARIA CICADAE Miquel), wherein,
(I) The monosaccharide composition of the anti-inflammatory polysaccharide comprises mannose, glucuronic acid, glucose and galactose;
(ii) The total sugar content of the anti-inflammatory active polysaccharide is >71%;
(iii) The molecular weight distribution range of the anti-inflammatory active polysaccharide is 1.3 multiplied by 10 2Da～2.93×105 Da;
(iv) The anti-inflammatory active polysaccharide has a large molecular weight of 2.93× 5 Da and a ratio of >78%.
As a preferred embodiment of the wheat bran-based anti-inflammatory active polysaccharide according to the present invention, wherein: the content of mannose in the monosaccharide composition of the anti-inflammatory active polysaccharide is 30-32%, the content of glucuronic acid is 9-10%, the content of glucose is 17-19%, and the content of galactose is 35-36%.
It is a further object of the present invention to provide the use of wheat bran-based anti-inflammatory active polysaccharides as active ingredient in the preparation of a medicament for the treatment of ulcerative colitis.
As a preferred scheme for the application of the wheat bran-based anti-inflammatory active polysaccharide as an active component in the preparation of a medicament for treating ulcerative colitis, the invention comprises the following steps: the ulcerative colitis is dextran sodium sulfate induced ulcerative colitis.
It is still another object of the present invention to provide the use of wheat bran-based anti-inflammatory active polysaccharides as functional ingredients in the preparation of food for protecting intestinal health.
The invention also aims to provide a preparation method of the anti-inflammatory active polysaccharide based on wheat bran, which comprises the steps of drying the wheat bran raw material, crushing and sieving, and adjusting the moisture content to 50% -60% to obtain a fermentation medium taking the wheat bran as a matrix;
Inoculating activated paecilomyces cicadae strain seed liquid to a sterilized fermentation medium, fermenting at constant temperature, collecting fermented wheat bran, drying, pulverizing, and sieving to obtain fermented wheat bran dry powder;
Leaching the fermented wheat bran dry powder by a hot water leaching method, rotating, concentrating, precipitating and centrifuging the extracting solution, collecting precipitate and freeze-drying to obtain fermented wheat bran crude polysaccharide;
removing protein from the fermented wheat bran crude polysaccharide by a Sevage method to obtain the anti-inflammatory active polysaccharide based on wheat bran.
As a preferred embodiment of the method for preparing the wheat bran-based anti-inflammatory active polysaccharide of the present invention, wherein: the paecilomyces cicadae is cultured and activated by an activation culture medium, wherein the activation culture medium comprises 1-8% of cicada pupa powder, 2% of glucose, 1.5% of agar, 1% of potato dipping powder, 0.03% of potassium dihydrogen phosphate, 0.015% of magnesium sulfate heptahydrate and 0.00008% of thiamine.
As a preferred embodiment of the method for preparing the wheat bran-based anti-inflammatory active polysaccharide of the present invention, wherein: the concentration of the paecilomyces cicadae seed solution is 10 6～108 cfu/mL.
As a preferred embodiment of the method for preparing the wheat bran-based anti-inflammatory active polysaccharide of the present invention, wherein: the inoculation amount of the paecilomyces cicadae seed liquid is 8-12% of that of wheat bran raw material.
As a preferred embodiment of the method for preparing the wheat bran-based anti-inflammatory active polysaccharide of the present invention, wherein: the fermentation temperature of the constant-temperature fermentation is 22-30 ℃ and the fermentation time is 3-10 d.
As a preferred embodiment of the method for preparing the wheat bran-based anti-inflammatory active polysaccharide of the present invention, wherein: the leaching temperature of the leaching treatment is 50-100 ℃ and the leaching time is 1-5 h.
The invention has the beneficial effects that:
(1) The invention utilizes the vigorous reproductive capacity and stronger bioconversion capacity of medicinal fungus paecilomyces cicadae, uses wheat bran as a fermentation substrate to carry out solid state fermentation, adopts a water extraction and alcohol precipitation method to obtain the fermented wheat bran polysaccharide with the total sugar content of 71.90 percent, the uronic acid content of 11.74 percent and the protein content of 3.45 percent from the fermented wheat bran, changes the monosaccharide composition of original wheat bran polysaccharide after the solid state fermentation of fungi, and converts the soluble sugar arabinoxylan in the original wheat bran into the galactomannan with the similar monosaccharide composition of the cordyceps cicadae polysaccharide.
(2) The active polysaccharide prepared by using the paecilomyces cicadae solid state fermentation wheat bran has good anti-inflammatory activity, and has more remarkable treatment effect on inhibiting colon shortening, spleen enlargement, intestinal tract injury and the rise of the expression level of pro-inflammatory cytokines TNF-alpha, 1L-1 beta and 1L-6.
(3) According to the invention, the medicinal fungus paecilomyces cicadae is selected as a solid fermentation strain, and wheat processing byproducts bran is used as a fermentation substrate, so that on one hand, the comprehensive utilization rate of wheat bran is effectively improved, and on the other hand, scientific support is provided for the development and utilization of fungus resources.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the description of the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art. Wherein:
FIG. 1 is a liquid chromatogram of WBP and FWBP molecular weight distribution detection in the practice of the present invention.
FIG. 2 is a liquid chromatogram of the detection of WBP and FWBP monosaccharide composition in the practice of the present invention.
FIG. 3 is a graph showing the effect of WBP and FWBP on sodium polysaccharide sulfate (DSS) induced remission of mouse colitis in the practice of the present invention.
FIG. 4 is a graph comparing the colon histopathological scores of mice induced by WBP and FWBP on sodium glycan sulfate (DSS) in the practice of the present invention.
FIG. 5 is a graph showing the inhibition of sodium sulfate glycan (DSS) -induced inflammatory factors in colon tissue of mice by WBP and FWBP in the practice of the present invention.
Detailed Description
In order that the above-recited objects, features and advantages of the present invention will become more apparent, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present invention is not limited to the specific embodiments disclosed below.
Further, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic can be included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
The raw materials used in the invention are all common commercially available analytical pure in the field without special description.
Specifically, the wheat bran used in the present invention is produced from developed flour mill;
The strain used in the invention is cordyceps sobolifera (ISARIA CICADAE Miquel) purchased from Beijing Beidou Nakazaku biological technology institute, and the strain number is 116724.
Example 1
The embodiment provides a preparation method of anti-inflammatory active polysaccharide based on wheat bran, which specifically comprises the following steps:
1) Activating strains: inoculating the inclined plane strain of the paecilomyces cicadae (ISARIA CICADAE Miquel) into an activation culture medium, and culturing for 5 days at 27 ℃ to obtain an activated cordyceps cicadae mycelium strain, wherein the formula of the paecilomyces cicadae activation culture medium comprises 6% of cordyceps cicadae pupa powder, 1.5% of agar, 1% of potato dipping powder, 0.03% of potassium dihydrogen phosphate, 0.015% of magnesium sulfate heptahydrate, 2% of glucose and 0.00008% of thiamine.
2) Seed liquid culture: 6 pieces of cordyceps sobolifera mycelium blocks after activation are picked up under the aseptic condition and inoculated into a conical flask filled with 100mL of culture medium, the conical flask is placed in a shaking table with the constant temperature of 27 ℃, the shaking table rotates at 150r/min, and paecilomyces cicadae seed liquid is obtained by filtering under the aseptic condition after shaking culture for 3 days.
3) Fermentation medium: drying wheat bran at 105 ℃ to constant weight, crushing and sieving with a 40-mesh sieve to obtain wheat bran dry powder, regulating the moisture content to 50wt% to obtain a fermentation culture medium taking wheat bran as a matrix, sterilizing the fermentation culture medium at 121 ℃ for 20min, and cooling to room temperature for later use;
4) Solid state fermentation: inoculating the seed liquid prepared in the step 2) into a sterilized fermentation medium according to 10% (w/v) of the mass of the wheat bran raw material, fermenting for 6 days in a constant temperature incubator at 27 ℃, collecting fermented wheat bran after fermentation, drying for 12 hours in a blast drying oven at 45 ℃, crushing and sieving with a 40-mesh sieve to obtain fermented wheat bran dry powder.
5) Polysaccharide extraction: and (3) extracting the fermented wheat bran polysaccharide from the fermented wheat bran dry powder prepared in the step (4) by adopting a hot water extraction method, wherein the extraction temperature is 80 ℃ and the extraction time is 2 hours. Concentrating the extractive solution at 60deg.C, adding 95% (v/v) ethanol solution to 80% ethanol mass concentration, precipitating at 4deg.C for 12 hr, centrifuging at 4000r/min for 10min, collecting precipitate, and lyophilizing to obtain fermented testa Tritici crude polysaccharide with a yield of 12.19%.
6) Polysaccharide deproteinization: removing protein from the fermented wheat bran crude polysaccharide in the step 5) by using a Sevage method, wherein the mass-volume ratio is 1:1, adding the fermented wheat bran polysaccharide into distilled water, and then adding the distilled water into the distilled water according to the volume ratio of 5:1, mixing a fermented wheat bran polysaccharide water solution with a Sevage solution, wherein the mass ratio of chloroform to n-butanol in the Sevage solution is 5:1, standing for 20min, centrifuging to remove the organic layer, removing protein from the upper polysaccharide solution again until no white substance is separated out between the organic phase and the water phase during standing, concentrating under reduced pressure at 60 ℃ to remove chloroform and n-butanol, lyophilizing to obtain fermented wheat bran polysaccharide with protein removed, and naming the fermented wheat bran polysaccharide as FWBP.
Comparative example 1
The comparative example is compared with wheat bran polysaccharide which is not fermented, and specifically comprises the following steps:
1) Polysaccharide extraction: drying wheat bran at 105 ℃ to constant weight, crushing and sieving with a 40-mesh sieve to obtain wheat bran dry powder. Extracting testa Tritici polysaccharide by hot water extraction at 80deg.C for 2 hr. Concentrating the extractive solution at 60deg.C, adding 95% (v/v) ethanol solution to 80% ethanol mass concentration, precipitating at 4deg.C for 12 hr, centrifuging at 4000r/min for 10min, collecting precipitate, and lyophilizing to obtain testa Tritici crude polysaccharide with a yield of 3.24%.
2) Polysaccharide deproteinization: removing protein from the wheat bran crude polysaccharide in the step 1) by using a Sevage method, wherein the mass-volume ratio is 1:1, adding wheat bran polysaccharide into distilled water, and then adding the wheat bran polysaccharide into distilled water according to a volume ratio of 5:1, mixing an aqueous wheat bran polysaccharide solution with a Sevage solution, wherein the mass ratio of chloroform to n-butanol in the Sevage solution is 5:1, standing after shaking for 20min, centrifuging to remove an organic layer, removing protein again from the upper polysaccharide solution until no white substances are separated out between an organic phase and a water phase after standing, concentrating under reduced pressure at 60 ℃ to remove chloroform and n-butanol, freeze-drying to obtain wheat bran polysaccharide after removing protein, and naming the wheat bran polysaccharide as WBP.
The polysaccharides of example 1 and comparative example 1 were analyzed as follows;
The total sugar content was measured using the phenol-sulfuric acid method, with a standard curve of y=9.3478x+0.0153, and r 2 = 0.9952; the uronic acid content is determined by m-hydroxybiphenyl method, the standard curve is y=20.96 x-0.0908, and r 2 = 0.9988; protein content was determined using coomassie brilliant blue method, standard curve y= 6.8871x-0.0034, r 2 =0.9987. The total sugar, uronic acid and protein content of the raw wheat bran polysaccharide WBP and the fermented wheat bran polysaccharide FWBP are shown in table 1.
Tables 1 WBP and FWBP Total sugar, uronic acid and protein content
Molecular weight distribution determination:
Molecular weight (Mw) was determined using an LC-20AT high performance liquid chromatography system, combined with a Rad-10A refractive index detector and OHpak SB-805HQ chromatography column. The mobile phase was ultrapure water at a flow rate of 0.8mL/min. The column temperature was maintained at 30 ℃. Polysaccharide samples (1 mg/mL) were dissolved in ultrapure water and filtered through a 0.22 μm membrane. The sample loading was 20. Mu.L.
FIG. 1 is a molecular weight distribution diagram of the fermented wheat bran polysaccharide FWBP and the raw wheat bran polysaccharide WBP.
As can be seen from FIG. 1, the liquid chromatograms of both the fermented wheat bran polysaccharide FWBP and the raw wheat bran polysaccharide WBP show inhomogeneities, the weight average molecular weight of the fermented wheat bran polysaccharide FWBP ranges from 1.3X10 2Da～2.93×105 Da, and the weight average molecular weight of the raw wheat bran polysaccharide WBP ranges from 1.7X10 2Da～2.98×105 Da. The wheat bran polysaccharide has reduced small molecular weight sugar content and increased large molecular weight content from 8.66% to 78.08%.
Monosaccharide composition analysis:
The monosaccharide composition of the wheat bran polysaccharide was determined using a 1-phenyl-3-methyl-5-pyrazolone (PMP) pre-column derivatization method.
Taking 5mg of wheat bran polysaccharide sample, adding 2mol/L trifluoroacetic acid into a sealed ampoule bottle, hydrolyzing for 6 hours at 120 ℃, evaporating to dryness under reduced pressure, performing derivatization reaction for 40 minutes before PMP column, extracting with chloroform for three times to remove excessive PMP, filtering the water phase with a 0.22 mu m filter membrane, and performing analysis by using a high performance liquid chromatograph. Agilent 1260 Infinicity II liquid chromatography was equipped, using Agilent ZORBAX SB-C18 column (4.6X105 mm,5 μm), mobile phase 0.1mol/L phosphate (pH=6.7) and acetonitrile (83:17), sample injection 10. Mu.L, flow rate set to 1.0mL/min, UV detection at 30deg.C, 250nm wavelength. The same applies to standard monosaccharides (Glc, man, gal, rha, ara, xyl, glcA and GalA). The monosaccharide composition of FWBP and WBP was determined by comparison with the monosaccharide standard retention time.
FIG. 2 is a diagram of the monosaccharide composition of the fermented wheat bran polysaccharide FWBP, the raw wheat bran polysaccharide WBP, and the mixed standard, wherein Man is mannose, rha is rhamnose, glcA is glucuronic acid, galA is galacturonic acid, glc is glucose, gal is galactose, xyl is xylose, and Ara is arabinose.
As can be seen from fig. 2, the composition of the wheat bran polysaccharide monosaccharides has been significantly changed after fermentation with paecilomyces cicadae, WBP is mainly composed of glucose (34.33%), xylose (27.26%) and arabinose (30.26%), while FWBP is mainly composed of mannose (31.93%), glucuronic acid (9.50%), glucose (17.94%) and galactose (35.55%).
Physical and chemical property analysis:
physical and chemical properties of wheat bran polysaccharide WBP and FWBP before and after fermentation were measured, including water holding capacity, oil holding capacity and swelling capacity, and the results are shown in Table 2.
TABLE 2 physicochemical Properties of wheat bran polysaccharide WBP and FWBP before and after fermentation
It can be seen that the polysaccharide FWBP after fermentation has a higher water retention, oil retention and swelling than the raw wheat bran polysaccharide WBP.
Example 2
This example differs from example 1 in that the culture medium formulation for strain activation was adjusted, specifically:
2% of cicada pupa powder, 1.5% of agar, 1% of potato soaked powder, 0.03% of monopotassium phosphate, 0.015% of magnesium sulfate heptahydrate, 2% of glucose and 0.00008% of thiamine;
The rest of the steps refer to example 1, and the wheat bran polysaccharide of this example is obtained by fermentation and is named FWBP1.
Example 3
This example differs from example 1 in that the culture medium formulation for strain activation was adjusted, specifically:
4% of cicada pupa powder, 1.5% of agar, 1% of potato soaked powder, 0.03% of monopotassium phosphate, 0.015% of magnesium sulfate heptahydrate, 2% of glucose and 0.00008% of thiamine;
The rest of the procedure is described in example 1, and the wheat bran polysaccharide of this example is obtained by fermentation and designated FWBP.
Example 4
This example differs from example 1 in that the culture medium formulation for strain activation was adjusted, specifically:
8% of cicada pupa powder, 1.5% of agar, 1% of potato soaked powder, 0.03% of monopotassium phosphate, 0.015% of magnesium sulfate heptahydrate, 2% of glucose and 0.00008% of thiamine;
The rest of the procedure is described in example 1, and the wheat bran polysaccharide of this example is obtained by fermentation and designated FWBP.
In vivo anti-inflammatory test
Experimental animals and feeding conditions: 80 SPF-class male Kunming mice, 19-21 g in body weight, 6 weeks old, were purchased from SPF Biotechnology Co., ltd (Beijing, china, production license: SCXK (Beijing) 2019-0010). 23+/-2 ℃ and 50% -60% relative humidity in a cage. Water and standard food are freely available. All animal experiments were performed according to the legislative route and ethical guidelines of the people's republic of China and approved by the animal care ethical committee of the university of Tianjin science and technology.
Experimental design and measurement index: after one week of adaptive feeding, all mice were randomly divided into 8 groups of 10 mice each. Normal drinking water was given to the normal group, and 3% dss water was given to the model group, the positive group and the polysaccharide treatment group for 7 days.
After the molding period is finished, the normal group mice and the model group mice are perfused with 0.2mL of sterile physiological saline. Mice in the positive group were given sulfasalazine (SASP) (200 mg/kg/day). Polysaccharide solutions administered separately in treatment groups, WBP administered WBP (200 mg/kg/day); group FWBP was given FWBP (200 mg/kg/day); group FWBP to FWBP1 (200 mg/kg/day); group FWBP was given FWBP2 (200 mg/kg/day); group FWBP was given FWBP3 (200 mg/kg/day).
The mice were perfused once daily for a recovery period of 7 days and their body weights were measured daily. Mice were sacrificed on day 21 of the experiment, spleens were weighed, colon length was measured, and a section of colon was taken for HE staining, intestinal damage was observed, and histomorphometric scoring was performed, and colon tissue of mice was collected for determining inflammatory factor levels in mice.
Data analysis: all experiments were performed after at least 3 independent experiments, and data were expressed as mean ± standard deviation. Analysis of data single factor analysis of variance was performed using SPSS21.0 software followed by Duncan test.
FIG. 3 is a graph of the remission of fermented wheat bran polysaccharide FWBP to sodium dextran sulfate (DSS) induced ulcerative colitis mice. As can be seen from fig. 3, the mice in the model group showed symptoms of weight loss, diarrhea, hematochezia, etc., indicating that the DSS-induced colitis mice model was successfully established.
As shown in fig. 3A, the body weight of the control mice tended to rise gradually over the course of the experiment. In contrast, DSS-modeled model groups showed a different degree of weight loss on day 5 and a gradual increase after FWBP administration on day 8, indicating that FWBP treatment enhanced weight recovery in DSS-induced colitis models.
Disease Activity Index (DAI) is a major indicator for evaluation of the symptoms of colitis in mice. Along with the duration of DSS induction, mice developed clinical symptoms such as weight loss, diarrhea, hematochezia, etc. As shown by DAI score (fig. 3B), the control group had no significant weight loss nor any clinical symptoms, and the DAI score was 0. DSS induction significantly increased DAI levels in mice compared to control. On days 8-14, with FWBP, WBP and sulfasalazine interventions, at the end of the intervention, the DAI scores were lower for mice in both FWBP and SASP groups than in the model group, indicating that FWBP intervention improved weight loss and reduced presence of diarrhea and bloody stool.
Colonic shortening is one of the typical symptoms of colitis. The colon length of each group of mice is shown in figure 3C, and the colon length of the model group of mice after DSS treatment is significantly shortened (p < 0.05) compared to the colon length of the control group. The effect of SASP, WBP, FWBP, FWBP, FWBP, FWBP3 on improvement of colon shortening was all significant (p < 0.05) compared to the model group, with FWBP group improving best.
Splenomegaly is one of the clinical symptoms of colitis patients. DSS-induced colitis mice develop splenomegaly. As shown in fig. 3D, the spleen index of DSS group was significantly higher than that of Con group, and treatment with SASP and polysaccharide inhibited the spleen index increase in inflammatory mice to some extent, and contrast found that FWBP group of treatment group inhibited the effect best.
FIG. 4 is a graph of colon injury and histological evaluation of polysaccharide FWBP prepared in example 1 on sodium dextran sulfate (DSS) induced ulcerative colitis mice.
As can be seen from fig. 4, the colon structure of the control mice is complete without obvious signs of injury or inflammation. DSS-induced model mice developed typical histological lesions of ulcerative colitis. Including inflammatory cell infiltration, crypt structure loss, and epithelial cell damage. However, both SASP and wheat bran WBP treatment before and after fermentation reduced colonic inflammatory cell infiltration and colonic structural damage, increased colonic goblet cells, and significantly reduced histopathological scores. In contrast, FWBP exhibited better therapeutic effect, the crypt structure was more complete, approaching the normal mouse state.
FIG. 5 is a graph showing the effect of different treatment groups on the level of inflammatory factors in colon tissue of mice induced by Dextran Sodium Sulfate (DSS). To demonstrate the effect of wheat bran polysaccharides on the inflammatory levels of mice with colon inflammation before and after fermentation, we examined the expression of major inflammatory factors in colon tissue of mice. As shown in FIG. 5, the expression of the pro-inflammatory cytokines TNF-. Alpha., IL-6, IL-1β was significantly increased in the model group compared to the Con group (p < 0.05). SASP group and polysaccharide treatment group treatment reduced TNF- α, IL-6 and IL-1β levels (FIGS. 5A-C). Wherein, the treatment of the fermented wheat bran polysaccharide FWBP has obviously better inhibition effect on the pro-inflammatory cytokines than WBP group (p < 0.05), compared with therapeutic SASP, FWBP has better inhibition effect. The anti-inflammatory cytokine IL-10 is responsible for maintaining immune homeostasis and thus inhibiting the progression of colitis. As shown in fig. 5D, the polysaccharide treated group significantly increased the level of IL-10 in the colon of DSS-induced mice (p < 0.05) compared to the model group, with FWBP group having a more pronounced recovery effect (p < 0.05) compared to the other treated groups. These results show that post-fermentation wheat bran polysaccharide FWBP exhibits superior therapeutic bioactivity for ulcerative colitis compared to the raw wheat bran polysaccharide WBP.
In conclusion, the invention utilizes the vigorous reproductive capacity and stronger bioconversion capacity of medicinal fungus paecilomyces cicadae, uses wheat bran as a fermentation substrate to carry out solid state fermentation, adopts a water extraction and alcohol precipitation method to obtain the fermented wheat bran polysaccharide with the total sugar content of 71.90 percent, the uronic acid content of 11.74 percent and the protein content of 3.45 percent from the fermented wheat bran, changes the monosaccharide composition of raw wheat bran polysaccharide after the solid state fermentation of fungi, and converts the soluble arabinoxylan in the raw wheat bran into the galactomannan with the similar monosaccharide component of the cordyceps sobolifera polysaccharide.
The active polysaccharide prepared by using the paecilomyces cicadae solid state fermentation wheat bran has good anti-inflammatory activity, and has more remarkable treatment effect on inhibiting colon shortening, spleen enlargement, intestinal tract injury and the rise of the expression level of pro-inflammatory cytokines TNF-alpha, 1L-1 beta and 1L-6.
According to the invention, the medicinal fungus paecilomyces cicadae is selected as a solid fermentation strain, and wheat processing byproducts bran is used as a fermentation substrate, so that on one hand, the comprehensive utilization rate of wheat bran is effectively improved, and on the other hand, scientific support is provided for the development and utilization of fungus resources.
It should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered in the scope of the claims of the present invention.
Claims (10)
1. An anti-inflammatory active polysaccharide based on wheat bran, characterized in that: the anti-inflammatory active polysaccharide is prepared by solid state fermentation of wheat bran by using paecilomyces cicadae (IsariacicadaeMiquel), wherein,
(I) The monosaccharide composition of the anti-inflammatory polysaccharide comprises mannose, glucuronic acid, glucose and galactose;
(ii) The total sugar content of the anti-inflammatory active polysaccharide is >71%;
(iii) The molecular weight distribution range of the anti-inflammatory active polysaccharide is 1.3 multiplied by 10 2Da～2.93×105 Da;
(iv) The anti-inflammatory active polysaccharide has a large molecular weight of 2.93× 5 Da and a ratio of >78%.
2. The wheat bran-based anti-inflammatory active polysaccharide of claim 1, wherein: the content of mannose in the monosaccharide composition of the anti-inflammatory active polysaccharide is 30-32%, the content of glucuronic acid is 9-10%, the content of glucose is 17-19%, and the content of galactose is 35-36%.
3. Use of an anti-inflammatory active polysaccharide according to claim 1 as active ingredient in the preparation of a medicament for the treatment of ulcerative colitis, characterized in that: the ulcerative colitis is dextran sodium sulfate induced ulcerative colitis.
4. Use of an anti-inflammatory active polysaccharide according to claim 1 as a functional ingredient in the preparation of a food product for protecting intestinal health.
5. The method for preparing an anti-inflammatory active polysaccharide according to claim 1, wherein: comprising the steps of (a) a step of,
Drying wheat bran raw materials, crushing, sieving, and adjusting the moisture content to 50% -60% to obtain a fermentation culture medium taking wheat bran as a matrix;
Inoculating activated paecilomyces cicadae strain seed liquid to a sterilized fermentation medium, fermenting at constant temperature, collecting fermented wheat bran, drying, pulverizing, and sieving to obtain fermented wheat bran dry powder;
Leaching the fermented wheat bran dry powder by a hot water leaching method, rotating, concentrating, precipitating and centrifuging the extracting solution, collecting precipitate and freeze-drying to obtain fermented wheat bran crude polysaccharide;
removing protein from the fermented wheat bran crude polysaccharide by a Sevage method to obtain the anti-inflammatory active polysaccharide based on wheat bran.
6. The method for preparing the anti-inflammatory active polysaccharide based on wheat bran according to claim 4, wherein: the paecilomyces cicadae is cultured and activated by an activation culture medium, wherein the activation culture medium comprises 1-8% of cicada pupa powder, 2% of glucose, 1.5% of agar, 1% of potato dipping powder, 0.03% of potassium dihydrogen phosphate, 0.015% of magnesium sulfate heptahydrate and 0.00008% of thiamine.
7. The method for preparing the anti-inflammatory active polysaccharide based on wheat bran according to claim 4, wherein: the concentration of the paecilomyces cicadae seed solution is 10 6～108 cfu/mL.
8. The method for preparing the wheat bran-based anti-inflammatory active polysaccharide according to any one of claims 4 or 5, wherein: the inoculation amount of the paecilomyces cicadae seed liquid is 8-12% of that of wheat bran raw material.
9. The method for preparing the anti-inflammatory active polysaccharide based on wheat bran according to claim 4, wherein: the fermentation temperature of the constant-temperature fermentation is 22-30 ℃ and the fermentation time is 3-10 d.
10. The method for preparing the anti-inflammatory active polysaccharide based on wheat bran according to claim 4, wherein: the leaching temperature of the leaching treatment is 50-100 ℃ and the leaching time is 1-5 h.
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